U.S. patent application number 11/446828 was filed with the patent office on 2006-12-21 for novel miniature inverted-repeat transposable elements (mites)-like element and transcriptional activation element.
Invention is credited to Takashi Fukuda, Takatoshi Koda, Mikiko Oyanagi, Yoshihiro Ozeki.
Application Number | 20060288438 11/446828 |
Document ID | / |
Family ID | 27328620 |
Filed Date | 2006-12-21 |
United States Patent
Application |
20060288438 |
Kind Code |
A1 |
Ozeki; Yoshihiro ; et
al. |
December 21, 2006 |
Novel miniature inverted-repeat transposable elements (MITEs)-like
element and transcriptional activation element
Abstract
The invention provides novel, carrot-derived MITE-like elements
(transposable elements). It further provides transcriptional
activation elements comprising at least one transposable element,
in particular one of the above MITE-like elements. Specifically, it
provides transcriptional activation elements having a DNA
comprising the nucleotide sequence shown under SEQ ID NO:1 or a
functional equivalent thereto and/or a DNA comprising the
nucleotide sequence shown under SEQ ID NO:2 or a functional
equivalent thereto. The transcriptional activation elements of the
invention can increase or activate the reduced expression of a
foreign gene introduced by the transgenic technology. Therefore,
the transcriptional activation elements contribute to stable
expression of a foreign gene introduced in a plant genome and are
useful in stably producing genetically modified plants.
Inventors: |
Ozeki; Yoshihiro; (Tokyo,
JP) ; Fukuda; Takashi; (Sagamihara-shi, JP) ;
Oyanagi; Mikiko; (Tokyo, JP) ; Koda; Takatoshi;
(Osaka, JP) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
27328620 |
Appl. No.: |
11/446828 |
Filed: |
June 5, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10031818 |
Mar 6, 2002 |
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PCT/JP00/04837 |
Jul 19, 2000 |
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11446828 |
Jun 5, 2006 |
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Current U.S.
Class: |
800/278 ;
435/468 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8201 20130101 |
Class at
Publication: |
800/278 ;
435/468 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C12N 15/82 20060101 C12N015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 12, 2000 |
JP |
2000/175825 |
Jul 21, 1999 |
JP |
11/206320 |
Jul 21, 1999 |
JP |
11/206316 |
Claims
1-19. (canceled)
20. An isolated miniature inverted-repeat transposable element
(MITE)-like element consisting of a DNA having a nucleotide
sequence shown as SEQ ID NO:1.
21. An isolated transcriptional activation element comprising a
MITE-like element consisting of the following DNA (a) or (b) as a
transposable element: (a) a DNA having a nucleotide sequence shown
as SEQ ID NO:1; (b) a DNA having a nucleotide sequence not less
than 90% homologous with the nucleotide sequence shown as SEQ ID
NO:1, which has a size of not more than about 2 kb, contains
perfect or imperfect terminal inverted repeat sequences in each of
the 5' and 3' terminal regions, contains a plurality of repetitions
of sequences represented by the formula (1): XttgcaaY (wherein X
represents g or t and Y represents a or c) or the formula (2):
Zatgcaa (wherein Z represents t or a) in the terminal inverted
repeat sequences or the intermediate region between the terminal
inverted repeat sequences, a continuously or discontinuously
repeated manner, and is capable of causing duplication of the
target sequence: (A)nG(A)n [n being an integer of not less than 1]
at the site of insertion thereof in a genomic gene.
22. The isolated transcriptional activation element according to
claim 21, wherein the transposable element is a tandem coupling
product consisting of the following DNA (a) or (b): (a) a DNA
having a nucleotide sequence shown as SEQ ID NO: 1; (b) a DNA
having a nucleotide sequence not less than 90% homologous with the
nucleotide sequence shown as SEQ ID NO:1, which has a size of not
more than about 2 kb, contains perfect or imperfect terminal
inverted repeat sequences in each of the 5' and 3' terminal
regions, contains a plurality of repetitions of sequences
represented by the formula (1): XttgcaaY (wherein X represents g or
t and Y represents a or c) or the formula (2): Zatgcaa (wherein Z
represents t or a) in the terminal inverted repeat sequences or the
intermediate region between the terminal inverted repeat sequences,
a continuously or discontinuously repeated manner, and is capable
of causing duplication of the target sequence: (A)nG(A)n [n being
an integer of not less than 1] at the site of insertion thereof in
a genomic gene, and a MITE-like element consisting of the following
DNA (c) or (d): (c) a DNA having a nucleotide sequence shown as SEQ
ID NO:2; (d) a DNA having a nucleotide sequence not less than 90%
homologous with the nucleotide sequence shown as SEQ ID NO:2, which
has a size of not more than about 1 kb, contains a perfect or
imperfect terminal inverted repeat sequence in each of the 5' and
3' terminal regions, and is capable of causing duplication of TA at
the site of insertion thereof in a genomic gene.
23. The isolated transcriptional activation element according to
claim 21, wherein the transposable element consists of a DNA having
the nucleotide sequence shown as SEQ ID NO:3.
24. The isolated transcriptional activation element according to
claim 21, wherein the transposable element consists of a DNA having
the nucleotide sequence shown as SEQ ID NO:14.
25. A transgene expression cassette which comprises the
transcriptional activation element of any of claims 21 to 24, and a
DNA sequence operatively joined to the transcriptional activation
element.
26. The transgene expression cassette according to claim 25,
wherein the DNA sequence operatively joined to the transcriptional
activation element comprises a promoter and/or a terminator.
27. The transgene expression cassette according to claim 25, which
further comprises, as the DNA operatively joined to the
transcriptional activation element, a desired transgene sequence to
be expressed.
28. A plasmid comprising the transcriptional activation element of
any of claims 21 to 24.
29. A plasmid comprising the transgene expression cassette of claim
25.
30. A transgenic plant, which contains the transgene expression
cassette of claim 25.
31. The transgenic plant as claimed in claim 30, which is corn,
rice, wheat, lily, chrysanthemum, cotton, soybean, beet, potato or
carica papaya.
32. A method of increasing transformation efficiency for transgenic
plant cells comprising the step of transforming the plant cells
with the plasmid of claim 29.
33. The method of claim 32 further comprising regenerating the
transformed plant cells to produce a transgenic plant.
Description
TECHNICAL FIELD
[0001] The present invention relates to novel, plant-derived
transposable elements and, more particularly, to a MITE (miniature
inverted-repeat transposable element)-like sequence. More
specifically, the present invention relates to novel transposable
elements isolated from carrot (Daucus carota).
[0002] The invention further relates to novel transcriptional
activation elements containing the transposable elements. More
specifically, it relates to a transcriptional activation element
which, when inserted in a gene, is capable of promoting the
transcription of a gene around or in the vicinity of the site of
insertion thereof or suppressing the transcription of said gene
from being inactivated. The invention still further relates to a
transgenic plant harboring said transcriptional activation
element.
BACKGROUND ART
[0003] Transposable elements are found in the genomes of almost all
living organisms, without distinction between prokaryotes and
eukaryotes, or between animals and plants. It is known that these
transposable elements can move from a genomic gene to another and
inactivate the genomic gene upstream or downstream from the
sequence in which such an element has been inserted. Further, it
has been revealed that, in addition to such actions, transposable
elements cause various types of genomic reorganization (deletion,
inversion, duplication, etc.) and it has been reported that the
above fact leads to genome plasticity, which is important for the
evolution of living organisms and, in particular, that transposable
elements play an important role in the evolution of living
organisms for environmental adaptation in response to genomic
stresses (McClintock, Science 26: 792-801 (1984); Arber et al.,
FEMS Microb. Ecol., 15: 5-14 (1994)).
[0004] Transposable elements are roughly classified into three
types: DNA type (transposons and insertion sequences), RNA type
(retrotransposons) (Berg et al. (ed.), Mobile DNA (1989)), and
miniature inverted-repeat transposable elements (MITEs) belonging
to neither of the above two types (Wessler et al., Curr. Opin.
Genet. Dev. 5: 914-821 (1995)).
[0005] As for DNA type and RNA type transposable elements among
them, their actual transposition in the genome has been established
whereas, for MITEs, no reports have so far been made about
evidences of their transposition in the genome in spite of their
being very similar to DNA type ones.
[0006] Most of MITEs have been found out, by computer retrieval,
from databases of nucleotide sequences of genomic genes of various
living organisms as being elucidated by genome projects currently
in progress. The first discovery was the discovery in 1992 of the
Tourist family by Bureau et al. (Bureau et al., Plant Cell 4:
1283-1294 (1992)). Since then, various MITEs have been found in
plant genomes and in insect and animal genomes.
[0007] Generally, MITEs are defined as having such characteristics
as (1) their having a perfect or imperfect inverted repeat sequence
at each of both the 5'- and 3'-terminal regions' (similar in this
respect to DNA type transposable elements), (2) occurrence of a
target duplication-like sequence, of a sequence consisting of two
or more nuclueotides, like the one formed upon insertion of a DNA
type transposable element into a genomic DNA, on both the terminal
sides of the inverted repeat sequences, and (3) their generally
having a size of shorter than 2 kb (Wessler et al., Curr. Opin.
Genet. Dev. 5: 914-821 (1995)).
[0008] As those MITEs which have an open reading frame coding for a
transposase between both the terminal inverted repeat sequences,
like DNA type transposable elements, there are known only MITEs of
the IS630-Tc1/Mariner family found in molds and animals (Kachroo et
al., Mol. Gen. Genet. 245: 339-348 (1994); Smit et al., Proc. Natl.
Acad. Sci. USA 93: 1443-1448 (1996)). As far as plant-derived MITEs
are concerned, however, no such transposase-encoding open reading
frame has been discovered. Therefore, many points remain
unelucidated concerning the mechanisms of transposition of plant
MITEs and concerning their actions.
[0009] By the way, the gene transfer experiments so far made in
gene manipulation in higher living organisms are mostly of the
nuclear genome insertion type. This is because higher living
organisms have no equivalent to the plasmid in prokaryotes.
[0010] For such gene transfer by insertion into the nuclear genome,
there are available physical methods comprising introducing a
vector coupled with a desired gene for insertion into the nuclear
genome of a higher living organism by the particle gun, lipofection
or electroporation technique, and biological methods comprising
introducing said vectors harboring the genes once into a virus or
microorganism and then introducing the same into the nuclear genome
of a higher living organism by taking advantage of the DNA
transfer/insertion system which said virus or microorganism
has.
[0011] These physical and biological methods, however, each has a
problem in that the expression activity of the gene inserted in a
higher living organism varies from an individual to another, hence
is not constant. In many instances, this is caused by silencing due
to the position effect (Peach et al., Plant Mol. Biol. 17: 46-60
(1991)). Such the position effect is a phenomenon found in yeasts
and many other eukaryotes and it is known that while there is no
change in the nucleotide sequence of the inserted gene itself, the
expression activity thereof varies markedly depending on the site
of insertion of said gene on the chromosome and, in certain cases,
the expression of the gene is completely inactivated (gene
silencing) (Molecular Biology of the Cell, 3rd edition, 434-435
(1995)).
[0012] Supposed as the causes of such phenomenon of the gene
silencing are the fact that all genes in the nuclear genome of a
higher living organism are not uniformly transcribed and the fact
that active sites where the gene is actively transcribed and the
cryptic sites where transcription of the gene is silenced at all
are intermingled in the nuclear genome. It is known that a gene
inserted in a cryptic site cannot be transcribed at all due to
complete failure of proteins necessary for transcription to
approach the gene or due to a change in nucleosome structure or
heterochromatinization as resulting from methylation of the genomic
DNA in this site, with the result that inactivation of gene
expression (gene silencing) occurs (Ng et al., Curr. Opin. Genet.
Dev., 9: 158-163 (1999); Matzke et al., Curr. Opin. Plant Biol. 1:
142-148 (1999)).
[0013] It is further known that, even in the same organism, this
gene silencing depends on the state of cell differentiation, as
seen with X chromosome of manmal. Furthermore, it is known that
once gene silencing is caused by a presently unknown mechanism, it
is inherited by the offspring of the next generation resulting from
mating (Molecular Biology of the Cell, 3rd edition, 434-453
(1995)).
[0014] Meanwhile, when, in gene manipulation, a desired gene
(foreign gene) is introduced into higher animal cells by a physical
or biological method (other than the homologous recombination
method used for gene knockout), the site of insertion of the gene
in the cell nuclear genome differs among cells and it is
substantially impossible to control it artificially. Thus,
introduction of a foreign gene into cells of a higher living
organism results in formation, in an uncertain manner, of cells
containing the foreign gene in an active site of the nuclear genome
and cells containing the gene in a cryptic site of the genome.
[0015] In the case of plant, it is known that a foreign gene, once
introduced in a cryptic site, undergoes the influence of the
cryptic site, with the result that the expression of the foreign
gene markedly diminishes or becomes null and it is also known that
even when a foreign gene is inserted in an active site, the
expression of the foreign gene becomes unstabilized and diminishes
due to gene silencing as the growth progresses by repetitions of
cell division (Peach et al., Plant Mol. Biol. 17: 46-60
(1991)).
[0016] A main presumable cause of inactivation of the expression of
a foreign gene is the structure of the nuclear DNA in the
chromosome. An element participating in the structure of the
nuclear DNA is a MAR (matrix attachment region; also called SAR
(scaffold attachment region) sequence. This was found as a sequence
anchoring a nuclear genomic DNA to a nuclear matrix. In animals, it
was shown that when a MAR-containing chicken A element is ligated
to a foreign gene to be inserted followed by gene transfer, the
expression of the gene inserted increased (Stief et al., Nature
341; 343-345 (1989)). Later studies, however, revealed that the MAR
contributes to an increase in gene expression efficiency but it,
when alone, cannot counteract the position effect (Bonifer et al.,
Nculeic Acids Res. 22: 4202-4210 (1994); Poljak et al., Nucleic
Acids Res. 22: 4386-4394 (1994)). In plants as well, studies have
been made to investigate the effects of MARs using transformants.
However, any reproducible results have not been obtained as yet. It
is not considered that a MAR alone can counteract the position
effect (Meshi: Shokubutsu no Genome Science (Genome Science of
Plants), 153-160 (1996), published by Shujunsha).
[0017] Currently, genetically modified plants have been developed
as genetically modified foodstuffs. However, the most important
problem to be solved in developing them is the phenomenon of gene
silencing which causes inactivation of the expression of foreign
genes, as mentioned hereinabove. In developing genetically modified
plants, it is considered necessary, for avoiding the phenomenon of
gene silencing, to select, among a very large number of plant
individuals, plant individuals having a foreign gene inserted at a
site where gene silencing is caused as scarcely as possible and,
further, to select plant individuals in which gene silencing will
not occur even after numerous generation for many years. For such
selection, it is necessary to raise a large number of plant
individuals and repeat over a number of generations and, therefore,
not only much labor and time are required but also a vast area of
land is required, namely a soil area problem arises.
[0018] Therefore, as another strategy of avoiding gene silencing,
it is an urgent and most important problem in genetic engineering
of plants to develop a method of suppressing the position
effect-due gene silencing or activating the transcription of an
inserted foreign gene and, more specifically, develop the so-called
"transcriptional activation element" having either of these
effects.
DISCLOSURE OF INVENTION
[0019] As mentioned hereinabove, many points remain unelucidated as
to what functions plant MITEs have. As for the functions of MITEs,
the possibility is presumable of their contributing to the
activation of gene expression based on the finding obtained by
studies so far made that MITEs are found frequently in regions
upstream of promoters of various genes (Wessler et al., Curr. Opin.
Genet. Dev. 5: 914-821 (1995)). Further, since a number of MITEs
are found in the vicinity of a matric attachment region (MAR)
(Avramova et al., Nucleic Acids Res. 26: 761-767 (1998)), it is
also presumable that they have an important connection with the
structure of the genome.
[0020] Thus, MITEs are very interesting transposable elements
(insertion elements) from the scientific viewpoint in elucidating
the relation between the genomic structure and the gene expression,
which has not yet been exhaustively investigated. In view of the
possibility of MITEs, when incorporated in a genomic structure
after transposition between genomic genes, changing the genomic
structure and, as a result, controlling the gene expression, their
utility as factors stabilizing, or preventing inactivation of, the
gene expression activity on the occasion of foreign gene
introduction can also be expected.
[0021] Thus, in a first aspect, the present invention has for its
object to provide novel, plant-derived MITE-like elements, which
have scientific and industrial utility, as mentioned above.
[0022] Specifically, the invention relates, first of all, to the
novel MITE-like elements mentioned below under 1 to 5 (hereinafter,
such MITE-like elements are sometimes referred to also as "IS2
elements" for convenience): [0023] 1. A MITE-like element capable
of causing duplication of the target sequence: (A)nG(A)n [n being
an integer of not less than 1]; [0024] 2. A MITE-like element as
defined above under 1 which has a perfect or imperfect terminal
inverted repeat sequence in each of the 5' and 3' terminal regions;
[0025] 3. A MITE-like element as defined above under 1 or 2 which
contains a plurality of repetitions of at least one of the
nucleotide sequences represented by the formula (1): XttgcaaY
(wherein X represents g or t and Y represents a or c) or the
formula (2): Zatgcaa (wherein Z represents t or a); [0026] 4. A
MITE-like element as defined above under any of 1 to 3 which has,
as terminal inverted repeat sequences, a nucleotide sequence shown
under SEQ ID NO:1 in the 5' terminal region and a nucleotide
sequence shown under SEQ ID NO:2 in the 3' terminal region; and
[0027] 5. A MITE-like element comprising the nucleotide sequence
shown under SEQ ID NO:3.
[0028] The present invention further relates to the novel MITE-like
elements mentioned below under 6 and 7 (hereinafter, such MITE-like
elements are sometimes referred to also as "IS1 elements" for
convenience): [0029] 6. A MITE-like element which has, as terminal
inverted repeat sequences, a nucleotide sequence shown under SEQ ID
NO:4 in the 5' terminal region and a nucleotide sequence shown
under SEQ ID NO:5 in the 3' terminal region, and is capable of
causing duplication of the target sequence TA; and [0030] 7. A
MITE-like element comprising the nucleotide sequence shown under
SEQ ID NO:6.
[0031] In its second aspect, the present invention has for its
object to provide the so-called "transcriptional activation factor"
capable of suppressing the gene inactivation phenomenon called gene
silencing due to a position effect or capable of activating the
transcription of a gene located in the vicinity or marginal region
thereof.
[0032] Specifically, the invention relates to the following
transcriptional activation factors mentioned below under 8 to 12:
[0033] 8. A transcriptional activation factor containing at least
one transposable element; [0034] 9. A transcriptional activation
factor as defined above under 8, in which the transposable element
is a MITE-like element; [0035] 10. A transcriptional activation
element as defined above under 9, in which the transposable element
comprises a MITE-like element comprising the following DNA (a) or
(b): [0036] (a) a DNA having the nucleotide sequence shown under
SEQ ID NO:1; [0037] (b) a DNA capable of hybridizing with a DNA
having the above nucleotide sequence (a) under stringent conditions
and coding for a MITE-like element capable of causing duplication
of (A)nG(A)n [n being an integer of not less than 1] at the site of
insertion thereof, and/or a MITE-like element comprising the
following DNA (c) or (d): [0038] (c) a DNA having the nucleotide
sequence shown under SEQ ID NO:2; [0039] (d) a DNA capable of
hybridizing with a DNA having the above nucleotide sequence (c)
under stringent conditions and coding for a MITE-like element
capable of causing duplication of TA at the site of insertion
thereof; [0040] 11. A transcriptional activation element as defined
above under 9, in which the transposable element is a tandem
coupling product from a MITE-like element comprising the following
DNA (a) or (b): [0041] (a) a DNA having the nucleotide sequence
shown under SEQ ID NO:1; [0042] (b) a DNA capable of hybridizing
with a DNA having the above nucleotide sequence (a) under stringent
conditions and coding for a MITE-like element capable of causing
duplication of (A)nG(A)n [n being an integer of not less than 1] at
the site of insertion thereof, and a MITE-like element comprising
the following DNA (c) or (d): [0043] (c) a DNA having the
nucleotide sequence shown under SEQ ID NO:2; [0044] (d) a DNA
capable of hybridizing with a DNA having the above nucleotide
sequence (c) under stringent conditions and coding for a MITE-like
element capable of causing duplication of TA at the site of
insertion thereof. [0045] 12. A transcriptional activation element
comprising a DNA having the nucleotide sequence shown under SEQ ID
NO:3.
[0046] The present invention further relates to a cassette for
expression of a gene introduced which comprises the transcriptional
activation element mentioned above. Specifically, there may be
mentioned the cassettes mentioned below under 13 to 15: [0047] 13.
A cassette for expression, in a plant, of a gene introduced which
comprises the transcriptional activation element defined above
under any of 8 to 12, and a DNA sequence operatively joined to said
factor; [0048] 14. An introduced gene expression cassette as
defined above under 13, in which the DNA sequence operatively
joined to the transcriptional activation element comprises a
promoter and/or a terminator; [0049] 15. An introduced gene
expression cassette as defined above under 14 which further
comprises, as the DNA sequence operatively joined to the
transcriptional activation element, a desired introduced gene
sequence to be expressed.
[0050] The present invention further relates to a plasmid which
contains the transcriptional activation element mentioned
hereinabove (as an introduced gene expression cassette, for
instance) and to a transgenic plant harboring the transcriptional
activation element introduced therein by utilizing such
plasmid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 is a representation of the structure of an IS2
element, which is a MITE-like element according to the
invention.
[0052] FIG. 2 is a representation of the structure of an IS1
element, which is a MITE-like element according to the invention,
and of its terminal inverted repeat sequences and its inserted
duplicate sequence (TA in the underlined parts).
[0053] FIG. 3 is a schematic representation of a method of
constructing gDCPAL3-pro/SK.
[0054] FIG. 4 is a comparative representation of the structures of
the carrot PAL genes gDCPAL3 and gDCPAL4.
[0055] FIG. 5 is a representation of the results of comparison
between the nucleotide sequence of a MITE-like element (IS1
element) of the present invention and the nucleotide sequences of
the so far known Stowaway family (Bureau and Wessler, Plant Cell,
6: 907-917 (1994). Sequences indicated by white letters on a black
background are terminal inverted repeat sequences showing
homology.
[0056] FIG. 6 is a representation of the imperfect inverted repeat
sequences and target duplication sequence (AAAAGAAAA in the
underlined parts) terminally found in an IS2 element, which is a
MITE-like element of the invention.
[0057] FIG. 7 is a schematic representation of a method of
constructing IS1-35S/SK.
[0058] FIG. 8 is a schematic representation of a method of
constructing IS2-35S/SK.
[0059] FIG. 9 is a schematic representation of a method of
constructing IS12-35S/SK.
[0060] FIG. 10 Lis a schematic representation of a method of
constructing MU3-35S/SK.
[0061] FIG. 11 is a schematic representation of a method of
constructing pIS1-35S/AB35S, pIS2-35S/AB35S, pIS12-35S/AB35S and
pMU3-35S/AB35S.
[0062] FIG. 12 is a representation of the results of Example 3 (1)
in which a comparison was made among the numbers of regenerated
calli, on selection media containing kanamycin, from cultured
tobacco BY-2 cells transformed by introduction of the constructs
pIS1-35S/AB35S (IS1), pIS2-35S/AB35S (IS2), pIS12-35S/AB35S (IS12)
and pAB35S (35S) (control). In the figure, the upper and lower
graphs show the results obtained by using selection media
containing 100 .mu.g/ml and 300 .mu.g/ml of kanamycin,
respectively.
[0063] FIG. 13 is a representation of the results of Example 3 (2)
in which a comparison was made between the GUS activity of tobacco
calli (control) resulting from introduction of pAB35S (35S) (left
graph) and the GUS activity of tobacco calli resulting from
introduction of pIS12-35S/AB35S (IS12) (right graph).
BEST MODES FOR CARRYING OUT THE INVENTION
I. Novel MITE-Like Elements
[0064] The known MITESs so far discovered include not only the
Tourist and Stowaway families mentioned above but also the families
Castaway, Crackle, Emigrant, Explorer, Ditto, Gaijin, Krispie, Pop,
Snap, Wanderer, Wujin, Wukong, Wuneng and Xbr. While these MITEs
are structurally characterized by all having a perfect or imperfect
invereted repeat sequence in the terminal region, the inverted
repeat sequences quite differ in nucleotide sequence and length
among the MITEs. Further, the target duplication sequence in the
MITE is TAA for Tourist, TA for Stowaway, TAA for Castaway,
GTTGATAT for Crackle, TTA for Ditto, TA for Emigrant, ATT or TAG or
TGA or GGT or GTT or GAA for Gaijin, TTGAAC for Krispie, AAAACAAA
or AAAAAAAA for Pop, TTTTTTT for Snap, TTA or TAA for Wanderer, TA
or CATA for Wujin, TATA or TACA for Wukong, TTAA or TTAT for
Wuneng, and TTAA for Xbr (any definite target duplication sequence
has not been found for Explorer) (Bureau et al. Proc. Natl. Acad.
Sci. USA, 93: 8524-8529 (1996); Casacuberta et al., Plant J., 16:
79-85 (1998); Song et al., Mol. Gen. Genet., 258: 449-456 (1998);
Tu, Proc. Natl. Acad. Sci. USA, 94: 7475-7480 (1997); Unsal and
Morgan, J. Mol. Biol., 248: 812-823 (1995)).
[0065] On the other hand, the IS2 element, an embodiment of the
above-mentioned novel MITE-like element of the present invention
has, as the target duplication sequence, (A)nG(A)n [n being an
integer not less than 1], which is not found in any of the so far
known MITEs (insertion elements). In this respect, the IS2 element
can be said to be an insertion element belonging to a novel family
different from any of the so far known families.
[0066] As for the IS1 element, which is an embodiment of the novel
MITE-like element of the present invention, it has TA as the target
duplication sequence and thus can be said to be a novel MITE-like
element belonging to the known family Stowaway (Bureau, T. E. and
Wessler, S. R. , Stowaway: a new family of inverted repeat elements
associated with the genes of both monocotyledonous and
dicotyledonous plants. Plant Cell, 6: 907-16 (1994)).
[0067] In the following, these IS2 and IS1 elements are
described.
1. IS2 Element
[0068] The IS2 element is characterized by causing target
duplication of (A)nG(A)n in the genomic gene at the site of
insertion. The number n may be any integer not less than 1. While
it is not particularly restricted, it is specifically, for example
2 to 6, preferably 3 to 5, more preferably 4.
[0069] More specifically, the MITE-like element of the present
invention is a DNA having a size of not more than about 2 kb,
preferably about 0.2 to 2 kb, has repeat sequences reverse in
direction to each other (terminal inverted repeat sequences) in the
5' and 3' terminal regions thereof.
[0070] From such viewpoint, the IS2 element of the present
invention meets the requirements concerning the above-mentioned
three characteristics of MITEs (Wessler et al. Curr. Opin. Genet.
Dev. 5: 914-821 (1995)), namely (1) that they have a perfect or
imperfect inverted repeat sequence at each of the 5' and 3'
termini, (2) that a repeat sequence comprising two or more base
pairs is -found as a target duplicate sequence in the same
direction on both sides of the inverted repeat sequence of the gene
insertion site and (3) that their size is not more than 2 kb, hence
can be identified as a transposable element (insertion element,
MITE-like element) having a MITE-like sequence.
[0071] The IS2 element of the present invention is structurally
characterized by containing, in the nucleotide sequence thereof, at
least one nucleotide sequence represented by the formula (1):
XttgcaaY (wherein X represents g or t and Y represents a or c) (SEQ
ID NO: 7 to 10) or the formula (2): Zatgcaa (wherein Z represents t
or a) (SEQ ID NO: 11 to 12) in a continuously or discontinuously
repeated manner.
[0072] The positioning and number of such repeat sequences are not
particularly restricted but they may be contained in the terminal
inverted repeat sequences occurring in both the terminal regions of
the IS2 element or in an intermediate region occurring between said
terminal inverted repeated sequences.
[0073] The IS2 element of the present invention specifically
includes the ones which contain a plurality of repeat sequences
represented by the above formula (1) and (2) in the intermediate
region between the terminal inverted repeat sequences and a
plurality of repeat sequences represented by the formula (1) in the
terminal inverted repeat sequences, as shown in FIG. 1.
[0074] The terminal inverted repeat sequences which the IS2 element
of the present invention has need not be strictly complementary to
each other but the only requirement is that the 5' and 3' terminal
regions can hybridize with each other under stringent conditions
and, as a result, the IS2 element can have such a stem structure as
shown in FIG. 1. In this sense, the IS2 element of the present
invention includes not only those having perfect inverted repeat
sequences as the terminal inverted repeat sequences but also those
having imperfect inverted repeat sequences as the terminal inverted
repeat sequences.
[0075] As specific examples of the IS2 element according to the
present invention, there may be mentioned the ones which have, as
the terminal inverted repeat sequences, the nucleotide sequence
shown under SEQ ID NO:1 in the 5' terminal region and the
nucleotide sequence shown under SEQ ID NO:2 in the 3' terminal
region. As a more specific example of the IS2 element, there may be
mentioned the one having the nucleotide sequence shown under SEQ ID
NO:3. The IS2 element may have one or more nucleotides substituted,
added or deleted in the terminal inverted repeat sequences or in
the sequence occurring between said repeat sequences if the
resulting modifications remain functional equivalents substantially
having the function or activity of the IS2 element itself. The
MITE-like element of the present invention includes such functional
equivalents as well.
[0076] As preferred functional equivalents, there may be mentioned
the ones substantially having the function or activity of the IS2
element having the nucleotide sequence shown under SEQ ID NO:3, and
causing target duplication of (A)nG(A)n [n being an integer not
less than 1] at the site of insertion and capable of hybridizing
with the above IS2 element under stringent conditions. As
"stringent conditions", there may be mentioned the conditions in
1.times.SSC plus 0.1% (w/w) SDS at 50.degree. C. or above over a
period of 1 hour. As the functional equivalents, there may be
mentioned more specifically the ones not less than 70%, preferably
not less than 85%, more preferably not less than 90%, still more
preferably not less than 95% homologous in nucleotide sequence with
the IS2 element shown under SEQ ID NO:3.
2. IS1 Element
[0077] The IS1 element of the present invention brings about target
duplication of TA at the site of genomic gene insertion and is
characterized by having, as the terminal inverted repeat sequences,
the nucleotide sequence shown under SEQ ID NO:4 in the 5' terminal
region and the nucleotide sequence shown under SEQ ID NO:5 in the
3' terminal region. The IS1 element of the invention is
specifically a DNA having a size of not more than about 1 kb,
preferably about 100 bp to 500 bp. In the light of such facts, the
IS1 element of the invention can be defined as a MITE-like element,
like the IS2 element mentioned above.
[0078] As the IS1 element of the present invention, there may
specifically be mentioned the one having the structure shown in
FIG. 2. More specifically, there may be mentioned the one having
the nucleotide sequence shown under SEQ ID NO:6. The MITE-like
element having such nucleotide sequence may have one or more
nucleotides substituted, added or deleted in the terminal inverted
repeat sequences or in the sequence occurring between these repeat
sequences of the 5' and 340 terminal regions if the resulting
modifications remain functional equivalents substantially having
the function or activity of the MITE-like element itself. The
MITE-like element of the present invention includes such functional
equivalents as well.
[0079] Preferred as the functional equivalents are those which
substantially have the function or activity of the MITE-like
element (IS1 element) having the nucleotide sequence shown under
SEQ ID NO:6 and which are at least 85%, preferably at least 90%,
more preferably at least 95% homologous in nucleotide sequence with
said IS1 element.
[0080] The MITE-like elements (IS2 and IS1 elements) described
hereinabove all have been discovered from the carrot genome, more
specifically from the carrot phenylalanine ammonia-lyase gene, as
mentioned later herein, and can be isolated and recovered as
described later herein in the example section. The MITE-like
element of the present invention is not limited in origin provided
that it has the structure and characteristics mentioned
hereinabove.
[0081] It is generally pointed out that transposable elements, as
self-mechanisms of automodification of the plant genome itself,
might possibly contribute markedly to evolution of the organism
concerned and to environmental adaptation.
[0082] As regards the MITE-like element of the present invention,
it is unknown as to the mechanisms by which it functions in
relation to the self-mechanisms of automodification of plant
genomes. However, unlike the case of retrotransposons (McDonald,
BioScience 40: 183-191 (1990), such a fact that it has an enhancer
element therewithin and a gene promoter is activated by insertion
of said enhancer element as resulting from transposition of said
element is not found.
[0083] Therefore, the MITE-like element of the present invention
can be considered to highly possibly cause changes in genomic
structure upon insertion thereof in a plant genome and thereby
contribute to dynamic changes of the genomic structure, such as
changes in unwindability of the genomic DNA or in nucleosome
structure, by the mechanisms quite different from those of the so
far known enhancer elements.
[0084] With the MITE-like element of the present invention, it
becomes possible, by utilizing the above property, to control the
expression of a gene located in the vicinity of said element by a
technique different from the so far known techniques. It is
generally pointed out that when a foreign gene is inserted in a
cryptic site of a genomic DNA, the expression thereof is suppressed
or inactivated. Therefore, it is considered possible, by utilizing
the MITE-like element of the present invention and based on the
above properties, to invigorate or activate the reduced or
suppressed ability to be expressed of a foreign gene introduced by
transgenic technique. Accordingly, the MITE-like element of the
present invention is useful in constructing a transgene expression
cassette and a plasmid containing said cassette in stably creating
genetically engineered plants and also useful in stably creating
genetically engineered organisms capable of expression of the
transgene by utilizing said cassette and plasmid.
II. Transcriptional Activation Element
[0085] The present invention further relates to a transcriptional
activation element.
[0086] The transcriptional activation element so referred to herein
includes a element capable of promoting the transcription of a
group of genes located in the vicinity or marginal region of said
element as well as a element capable of inhibiting suppression of a
desired foreign gene introduced in a genomic DNA or the like from
being inactivated by gene silencing due to the position effect. The
so-far known elements (factors) in charge of transcriptional
activation each activates the transcription of a specific gene by
occurring, as an enhancer, in the vicinity of the promoter of said
gene and cis-acting directly on said promoter. On the contrary, the
transcriptional activation element of the present invention
promotes the transcription of a single gene or a group of a
plurality of genes located in the vicinity or marginal region
thereof, irrespective of position relative to the promoter and,
further, includes those substantially promoting the transcription
by suppressing the inherent phenomenon of transcription
inactivation, hence conceptually includes a broader range of
factors as compared with the prior art concept of transcriptional
activation element.
[0087] The transcriptional activation element of the present
invention is characterized by containing at least one transposable
element.
[0088] The transposable element so referred to herein includes all
of the above-mentioned DNA type and RNA type ones and MITEs. Thus,
the transcriptional activation element of the present invention
includes those containing at least one of these as a transposable
element, regardless of whether it is derived from the same species
or a different species.
[0089] A preferred transcriptional activation element contains a
MITE(s) as the transposable element.
[0090] While a MITE is defined, as mentioned above, as an element
having such characteristics as (1) having a perfect or imperfect
inverted repeat sequence in each of both 5' and 3' terminal regions
(similar in this respect to DNA type transposable elements), (2)
having, on both sides of the inverted repeat sequence, a target
duplication sequence comprising repeat sequences arranged in the
same direction and comprising two or more base pairs, like the ones
formed upon insertion of a DNA type transposable element in a
genomic DNA and (3) having a size generally of shorter than 2 kb
(Wessler et al. , Curr. Opin. Genet. Dev. 5: 914-821 (1995)), any
MITE belonging to such category of definition can be used as the
transposable element in carrying out the present invention. As
MITEs particularly suited among others, there may be mentioned the
above-mentioned novel MITE-like elements of the present invention,
namely the "IS2 element", "IS1 element", and functional equivalents
thereto.
[0091] Thus, the transcriptional activation element of the present
invention preferably contains at least one nucleotide sequence
selected from among said IS2 element, IS1 element, and functional
equivalents thereto.
[0092] More specifically, the transcriptional activation element of
the present invention includes, among others, (1) the one having
the nucleotide sequence of the IS1 element or a functional
equivalent thereof, (2) the one having the nucleotide sequence of
the IS2 element or a functional equivalent thereof, (3) the one
having a nucleotide sequence resulting from tandem joining of the
nucleotide sequence of the IS1 element or a functional equivalent
thereof and the nucleotide sequence of the IS2 element or a
functional equivalent thereof (the order of the IS1 element and IS2
element being arbitrary) and (3) the one having a nucleotide
resulting from joining of the nucleotide sequence of the IS1
element (or IS2 element) or a functional equivalent thereto and the
nucleotide sequence of the IS2 element (or IS1 element) or a
functional equivalent thereto via an arbitrary nucleotide sequence.
Preferred examples of the transcriptional activation factor, there
may be mentioned (i) the IS2 element or a functional equivalent
thereto and (ii) the product of tandem joining of the IS1 element
(or IS2 element) or a functional equivalent thereto and the IS2
element (or IS1 element) or a functional equivalent thereto. As a
specific example of the latter (ii), there may be mentioned the one
having the nucleotide sequence shown under SEQ ID NO:3.
[0093] Referring to the transcriptional activation element
mentioned above under (4), the intervening nucleotide sequence
between the IS1 element and IS2 element (the order being arbitrary)
is not particularly restricted but may be any nucleotide sequence
on condition that the effects of the invention are not
counteracted. As a specific, but nonlimitative, example, there may
be mentioned one derived from the carrot PAL promoter sequence.
Generally, such nucleotide sequence can have a size of 5 to 1,000
bp, preferably 300 to 500 bp. As a transcriptional activation
element of such mode of embodiment, there may be specifically
mentioned the one having the nucleotide sequence shown under SEQ ID
NO:14.
[0094] The transcriptional activation element of the present
invention can be operatively joined to a desired gene sequence to
be introduced into a plant body (transgene sequence (inclusive of
foreign gene sequence)) and, further, the transcriptional
activation element joined to said transgene sequence can be
operatively joined to a functional DNA sequence or sequences, such
as a promoter functional in a plant and/or a terminator functional
in a plant.
[0095] The expression "operatively joined" as used herein means
that the transcriptional activation element is located at a
position sufficiently close to the above-mentioned transgene
sequence or various functional DNA sequences to exert its influence
on these sequences, irrespective of insertion site and
direction.
[0096] The transgene to be used in the practice of the present
invention includes those DNAs which are desired to be expressed in
plants, whether homologous or heterologous to said plants. Such
transgene includes, but is not limited to, genes coding for
.beta.-glucuronidase; antibiotic resistance genes; genes coding for
insecticidal and bactericidal proteinaceous toxins; genes for
antipathogenic compounds; genes synthesizing hypersensitivity
compounds, such as peroxidases, glucanases, chitinases, and
phytoalexins; agrochemical, herbicide and microbicide resistance
genes; genes synthesizing plant enzymes (e.g. enzymes connected
with the contents and qualities of proteins, starch, saccharides
and fats) and genes for regulatory factors therefor; genes related
to plant enzyme inhibitors, such as protease and amylase
inhibitors; genes involved in plant hormone synthesis; genes
involved in insect hormone and pheromone synthesis; genes involved
in the synthesis of medicinal and nutritional compounds, such as
.beta.-carotene and vitamins; and antisense transcripts interfering
with nucleotide sequences occurring in plants (Transgenic Plant,
vol. 1, Academic Press, 1993).
[0097] The present invention further relates to a gene expression
cassette suited for application to plants, which comprises the
above transcriptional activation element and a DNA sequence or
sequences operatively joined thereto. The "gene expression
cassette" so referred to herein means a plasmid to be used for
introduction into plants as well as a subfragment thereof.
[0098] As the DNA sequence operatively joined to the
transcriptional activation element, there may be mentioned
functional DNA sequences such as promoter and terminator. Said DNA
sequence can include any of the above-mentioned transgene
sequences.
[0099] The promoter, so referred to herein, means a DNA sequence
which, when the structural gene for a desired protein is joined
thereto downstream from said promoter, can regulate the expression
of said protein via transcription followed by translation in plant
cells, and it includes all promoters functional in plants and used
in the relevant field of art for the transformation of plants. A
number of promoters have so far been used in transforming plants,
including, for example, the promoters isolated from Agrobacterium
tumefaciens, namely the octopine synthase (ocs) promoter (L. Comai
et al., 1985; C. Waldron et al., 1985), mannopine synthase (mas)
promoter and nopaline synthase (nos) promoter. The cauliflower
mosaic virus 35S promoter, which is generally used in the
transformation, can adequately be used in practicing the present
invention. Modifications of the 35S promoter, for example the two
parallel 35S promoters (R. Kay et al., 1987) and the mas-35S
promoters (L. Comai et al., 1990), can also be used. Furthermore,
the cauliflower mosaic virus 19S promoter (J. Paszkowski et al.,
1984) and scrophularia mosaic virus-derived 34S promoter (M. Sanger
et al., 1990) can also be included. As further examples, there may
be mentioned the actin promoter, ribulose-1,5-bisphosphate
carboxylase small subunit (rbcS) promoter and so forth, which are
plant-derived promoters.
[0100] The terminator includes DNA sequences capable of efficiently
terminating the transcription of a desired structural gene in
plants and includes all terminators functioning in plants that are
used in the relevant field of art for the transformation of plants.
Specifically, the nopaline synthase (nos) terminator, for example,
can be mentioned as a typical example.
[0101] The transcriptional activation element of the present
invention or the transgene expression cassette comprising said
element can be used for inducing or regulating the expression in a
plant of the gene introduced, and this widely applies to plants in
general.
[0102] The plant is not particularly restricted but includes, among
others, agriculturally useful plants, whether monocotyledonous or
dicotyledonous. Among the monocotyledonous plants, for instance,
there are corn, rice, wheat, barley, African or Indian millet, oat,
rye, millet and other cereal crop plants as well as lily, orchid,
iris, palm, tulip, sedge and other various ornamental plants. The
dicotyledons include chrysanthemum, snapdragon, carnation,
magnolia, poppy, cabbage, rose, pea, poinsettia, cotton, cactus,
carrot, cowberry, peppermint, sunflower, tomato, elm, oak, maple
tree, poplar, soybean, melon, beet, rape, potato, lettuce, carica
papaya, etc.
[0103] The present invention further provides a transgenic plant
which contains the transcriptional activation element of the
present invention or the transgene expression cassette of the
present invention comprising said element and in which the
phenomenon of the position effect-due gene silencing (inactivation)
has been suppressed for the desired foreign gene introduced or the
transcription of the foreign gene has been activated. Said
transgenic plant includes the offspring thereof.
[0104] The term "plant" as used herein is intended to include not
only a perfect plant body but also a portion of a plant body, such
as a leaf, seed, bulb or cutting. It further includes protoplasts,
plant calli, mericlones and like plant cells as well.
[0105] The method of producing such transgenic plant is not
particularly restricted but may be any of those DNA introduction
methods which are conventionally used in the relevant field of art.
Specifically, it is a method of introducing a DNA into plant cells
using an expression plasmid containing the transcriptional
activation element of the present invention or a transgene
expression cassette containing said element and includes, among
others, such known methods as the Agrobacterium method, electric
method (electroporation) and particle gun method.
[0106] The thus-obtained plant cells containing the transcriptional
activation element of the invention, the transgene expression
cassette containing said element, or the expression plasmid can be
regenerated by one of the conventional methods used in the plant
tissue culture technology as described, for example, in S. B.
Gelvin, R. A. Schilperoot and D. P. S. Verma: Plant Molecular
Biology Manual (1991), Kluwer Academic Publishers or Valvekens et
al., Proc. Natl. Acad. Sci., 85: 5536-5540 (1988), whereby plant
bodies, or portions thereof, derived from said plant cells can be
obtained.
[0107] The expression plasmid of the present invention may be any
one provided that it contains the desired DNA sequence to be
introduced (transgene) together with the transcriptional activation
element and functional DNA sequences such as a promoter and a
terminator. It is preferred, however, that these DNA sequences be
operatively joined to one another. The phrase "operatively joined"
means that the plasmid functions for the intended purpose.
Specifically, it is implied that when the plasmid in question is
introduced into plant cells, the desired transgene (structural
gene) is expressed under the control of the transcriptional
activation element and the expression is efficiently terminated by
the action of the terminator, without inactivation of the promoter
contained in said plasmid.
[0108] The present invention further includes a method of causing
expression of a transgene in a plant. Such method can be carried
out at least by the step of introducing, into a plant, the
transgene expression cassette with the transgene integrated
therein, such as mentioned above,and the step of causing expression
of said transgene in said plant. The introduction of the transgene
expression cassette (DNA) into the plant and the expression of said
transgene can both be effected by the techniques per se known in
the art (Plant Molecular Biology Manual, 1991, Kluwer Academic
Publishers).
EXAMPLES
[0109] The following examples illustrate the present invention in
further detail. They are, however, by no means limitative of the
scope of the present invention. The genetic engineering techniques
and the experimental procedures of molecular biology (restriction
enzyme treatment conditions, ligation reaction conditions,
transformation into Escherichia coli, etc.), which are to be
employed in the practice of the present invention, can be carried
out as generally and widely employed, for example as described in
J. Sambrook, E. F. Frisch, T. Maniatis: Molecular Cloning, 2nd
edition, Cold Spring-Harbor Laboratory Press, 1989 and D. M.
Glover: DNA Cloning, IRL Press, 1985, among others.
Example 1
1) Target Plant, Target Gene
[0110] In searching for MITE-like elements, carrot (Daucus carota
L. cv. Kurodagosun) was used as the target plant, and the
phenylalanine ammonia-lyase (PAL) of said carrot as the target
gene.
[0111] 2) Cloning of the carrot PAL genes gDCPAL3 and gDCPAL4
Carrot genomic sequences were cloned from a carrot genomic DNA
library. The carrot genomic DNA library was constructed, as
previously described by the present inventors (Ozeki, Y., Davies,
E. and Takeda, J.: Structure and expression of chalcone synthase
gene in carrot suspension cultured cells regulated by 2,4-D. Plant
Cell Physiol., 34: 1029-1037 (1993)), from cultured carrot cells
(Ozaki, Y. and Komamine, A.: Induction of anthocyanin synthesis in
relation to embryogenesis in a carrot suspension culture;
Correlation of metabolic differentiation with morphological
differentiation. Physiol. Plantarum, 53: 570-577 (1981)) using the
.lamda. EMBL3 vector (product of Toyobo).
[0112] Specifically, carrot genomic DNA was prepared from carrot
freeze-dried using a cetyltrimethylammonium bromide (CTAB) solution
according to the method of Murray and Thompson (1980) (Murray, M.
G. and Thompson, W. F.; Rapid isolation of high molecular weight
plant DNA. Nucl. Acids Res. 8: 4321-4325 (1980)). The genomic DNA
obtained was partially digested with Sau3AI and the digested DNA
was fractionated by size by the sucrose density gradient method.
The DNA fraction within 15 to 20 kbp was collected and ligated to
the BamHI-digested .lamda. EMBL3 vector, followed by packaging in
phage particles, to thereby construct a carrot nuclear library.
[0113] Then, the carrot genomic library was screened (Sambrook et
al. 1989) for carrot PAL genomic clones using the PAL cDNA (ANT-PAL
cDNA) as the probe cloned by the method of Ozeki et al. (Ozeki, Y.,
Matsui, K., Sakuta, M., Matsuoka, M. Ohashi, Y., Kano-Murakami, Y.,
Yamamoto, N, and Tanaka, Y.: Differential regulation of
phenylalanine ammonia-lyase genes during anthocyanin synthesis and
by transfer effect in carrot cell suspension cultures. Physiol.
Plantarum, 80: 379-387 (1990)). The hybridization for screening of
the carrot genomic library was effected by overnight treatment at
68.degree. C. with a solution containing 6.times.SSC, 60 mM sodium
phosphate (pH 6.8), 10 mM EDTA, 1% SDS, 0.02% polyvinylpyrrolidone,
0.02% Ficoll 400 and 100 .mu.g/ml denatured salmon sperm DNA. The
membrane washing was carried out twice (15 minutes.times.2) in a
2.times.SSC solution containing 0.5% SDS at room temperature, twice
(10 minutes.times.2) in a 0.1.times.SSC solution or a 1.times.SSC
solution containing 0.1% SDS at room temperature and, finally,
twice (30 minutes.times.2) in a 0.1.times.SSC solution at
68.degree. C.
[0114] As a result, eight positive clones were obtained.
Restriction enzyme maps were prepared for such clones and,
according to the maps, the clones were classifiable into two genes,
which were named gDCPAL3 and gDCPAL4, respectively.
[0115] With gDCPAL3, the .lamda. phage of a positive clone was
cultured, .lamda. DNA was extracted, cleaved with BamHI and
subjected to Southern transfer to a nylon membrane, according to
the methods described in Sambrook et al. (1989). With this,
Southern analysis was carried out using a probe prepared from the
above-mentioned ANT-PAL cDNA by cleavage with EcoRI and labeling a
DNA fragment (984 bp) thereof at the 5' end with [.sup.32P], and a
2.77 kbp DNA fragment hybridizing with said probe was obtained.
This DNA fragment was cleaved with BamHI and subcloned in the
pBluescript SK plasmid treated with calf intestine alkaline
phosphatase (CIP), to give gDCPAL3-pro/SK (cf. FIG. 3). Then, the
plasmid gDCPAL3-pro/SK obtained was cleaved with the restriction
enzymes SalI and ApaI, a series of deletion DNA fragment groups
were produced using exonuolease III and mung bean nuclease by the
method described by Sambrook et al. (1989), and the nucleotide
sequence of the gDCPAL3 DNA was determined using them. The site of
transcription initiation (+1) was determined based on the positions
of bands as found by the primer extension method using mRNA
extracted from carrot as described by Ozeki and Takeda (1994)
(Regulation of phenylalanine ammonia-lyase genes in carrot
suspension cultured cells. Plant Cell. Tissue and Organ Culture,
38: 221-225 (1994)).
[0116] With gDCPAL4, the plasmid gDCPAL4-pro/SK was produced and
the nucleotide sequence of the gDCPAL4 DNA corresponding to the
above was determined in the same manner. Specifically, the .lamda.
DNA obtained from the .lamda. phage of a positive gDCPAL4 clone was
cleaved with HindIII and BamHI, Southern analysis was performed
using the same probe as mentioned above, and a 1.63 kbp DNA
fragment hybridizing with the probe was cleaved with HindIII and
BamHI and subcloned in the pBluescript SK plasmid treated with CIP,
to give gDCPAL4-pro/SK. Then, the thus-obtained plasmid
gDCPAL4-pro/SK was cleaved with the restriction enzymes XbaI and
BstXI, a series of deletion DNA fragment groups were produced using
exonuclease III and mung bean nuclease by the method described by
Sambrook et al. (1989), and the nucleotide sequence of the gDCPAL4
DNA was determined using them.
3) Results
[0117] Comparison of the nucleotide sequences of gDCPAL3 and
gDCPAL4 revealed that the promoter region of gDCPAL3 has miniature
inverted-repeat transposable elements (MITEs) having imperfect
inverted repeat sequences not found in gDCPAL4 were present at two
sites, namely -1897 to -1599 (299 bp in length) and -1157 to -389
(769 bp in length) (FIG. 4). These sequences were named IS1 and
IS2, respectively.
[0118] These sequences and the nucleotide sequences around the
sites of insertion were sequenced using an autosequencer (product
of LICOR model 4000L). The nucleotide sequence of IS1 is shown
under SEQ ID NO:2 and the nucleotide sequence of IS1 under SEQ ID
NO:1.
[0119] The characteristics of these IS1 and IS2 were as
follows:
(1) IS1
[0120] It had the nucleotide sequence shown under SEQ ID NO:2
(total length: 299 bp), had inverted repeat sequences (32 bp),
which were imperfect to each other, in the 5' and 3' terminal
regions, and had a target duplication sequence, TA, at the site of
insertion into the genome serving as the target gene. Based on
these facts, it was estimated to be the gene sequence of a novel
MITE element belonging to the family Stowaway already reported
(Bureau, T. E. and Wessler, S. R. Stowaway: a new family of
inverted repeat elements associated with the genes of both
monocotyledonous and dicotyledonous plants. Plant Cell, 6: 907-16
(1994)). The stem structure of the element IS1 and the structure of
the terminal inverted repeat sequence region and of the insertion
site region are shown in FIG. 2.
[0121] Based on the nucleotide sequence information obtained,
homology analysis of the nucleotide sequence was performed using
commercial databases (e.g. GENE TYX-MAC/CD1995), whereupon, in the
terminal inverted repeat sequences, 70-90% homology was found with
the gene sequence of MITE elements belonging to the Stowaway family
(Bureau and Wessler (1994)). It was thus confirmed that said
element is a transposable element belonging to the Stowaway family
(FIG. 5).
(2) IS2
[0122] It had the nucleotide sequence shown under SEQ ID NO:1
(total length: 769 bp), had inverted repeat sequences (158 bp),
which were imperfect to each other, in the 5' and 3' terminal
regions, and had a target duplication sequence, AAAAGAAAA, at the
site of insertion into the genome serving as the target gene.
Homology comparison of the nucleotide sequence was made but no
homology with known transposable elements was detected. It was thus
found that it is a transposable element, particularly a MITE-like
element, constituting a novel family belonging to none of the so
far known transposable element families. The overall structure
(stem structure) of the IS2 element is shown in FIG. 1 and the
structure (nucleotide sequence) of its terminal inverted repeat
sequence region and of the insertion site region in FIG. 6.
Example 2
(1) Cloning of IS1, IS2, IS12 and MU3
(i) Cloning of IS1 (FIG. 7)
[0123] From among the plasmids having. a deletion DNA fragment
derived from the 3' terminus of the gDCPAL3 promoter region as
prepared for nucleotide sequence determination in Example 1, a
plasmid (gDCPAL3-IS1/SK) with deletion to -1581 was selected, and a
321 bp DNA fragment was excised by cleaving that plasmid with KpnI,
rendering blunt-ended using T4 DNA polymerase and cleaving with
ScaI, followed by agarose gel electrophoresis. This was subcloned
in the plasmid pBluescript SK cleaved with HincII and treated with
CIP. Plasmids were extracted from among Escherichia coli colonies
harboring a plurality of independent clones obtained by the above
subcloning and the nucleotide sequences thereof were determined for
revealing the direction of each DNA fragment inserted. In this way,
a plasmid with the 5' terminal side of IS1 being inserted on the
KpnI side of the multiple cloning site of the pBluescript SK
plasmid was selected and named IS1/SK. Further, the cauliflower
mosaic virus 35S promoter (35S) fragment obtained by cleavage of
pBI221 (Clontech Inc.) with HindIII and SmaI was recovered by
agarose gel electrophoresis and subcloned in the pBluescript SK
plasmid cleaved with HindIII and SmaI and treated with CIP, to give
a plasmid named 35S/SK. From IS1/SK, the insert DNA fragment was
excised by cleaving with KpnI and HindIII, followed by agarose gel
electrophoresis, and this was subcloned in the 35S/SK plasmid
cleaved with KpnI and HindIII and treated with CIP, to give
IS1-35S/SK having the IS1 region in the upstream region of the 35S
promoter region.
(ii) Cloning of IS2 (FIG. 8)
[0124] From among the plasmids having a deletion DNA fragment
derived from the 3' terminus of the gDCPAL3 promoter region as
prepared for nucleotide sequence determination in Example 1, a
plasmid (gDCPAL3-IS12/SK) showing deletion to -389 was selected,
and a 797 bp DNA fragment was excised by cleaving that plasmid with
KpnI, rendering blunt-ended using T4 DNA polymerase and cleaving
with DdeI, followed by agarose gel electrophoresis. This was
subcloned in the plasmid pBluescript SK cleaved with HincII and
treated with CIP. Plasmids were extracted from among E. coli
colonies harboring a plurality of independent clones obtained by
the above subcloning and the nucleotide sequences thereof were
determined for revealing the direction of each DNA fragment
inserted. In this way, a plasmid with the 5' terminal side of IS2
being inserted on the KpnI side of the multiple cloning site of the
pBluescript SK plasmid was selected and named IS2/SK-1. One with
the 3' terminal side of IS2 being inserted on the KpnI side, namely
in the reverse direction, was selected and named IS2/SK-2
(reverse).
[0125] IS2/SK-1 was cleaved with KpnI and HindIII and the insert
DNA fragment was recovered by agarose gel electrophoresis, and this
was subcloned in the 35S/SK plasmid cleaved with KpnI and HindIII
and treated with CIP, to give IS2-35S/SK having the IS2 region
(positive strand) in the upstream region of the 35S promoter
region.
(iii) Cloning of IS12 (IS1-IS2 Tandem Coupling Product) (FIG.
9)
[0126] The IS2/SK-2 (reverse) obtained as described above under
(ii) was cleaved with KpnI, rendered blunt-ended using T4 DNA
polymerase and then cleaved with HindIII, and the insert DNA
fragment was recovered by agarose gel electrophoresis. This was
cleaved with PstI, rendered blunt-ended using T4 DNA polymerase and
then subcloned in the IS1-35S/SK plasmid cleaved with HindIII and
treated with CIP, to give IS12-35S/SK having the IS1 region and IS2
region in the upstream region of the 35S promoter region in a
tandem manner.
(iv) Cloning of MU3 (FIG. 10)
[0127] As for MU3, gDCPAL3-IS12/SK was cleaved with KpnI, then
rendered blunt-ended using T4 DNA polymerase and cleaved with ScaI,
and a 1,514 bp DNA fragment was recovered by agarose gel
electrophoresis. This was subcloned in the pBluescript SK plasmid
cleaved with HincII and treated with CIP. Plasmids were extracted
from a plurality of E. coli colonies harboring each independent
clone as obtained by the above subcloning and the nucleotide
sequences thereof were determined to thereby check the direction of
DNA fragment insertion. A plasmid with the 5' terminal side of IS1
inserted on the KpnI side of the multicloning site of the
pBluescript SK plasmid was selected and named MU3/SK. The insert
DNA fragment was excised by cleaving MU3/SK with KpnI and HindIII,
followed by agarose gel electrophoresis. This was subcloned in the
35S/SK plasmid cleaved with KpnI and HindIII and treated with CIP,
to give MU3-35S/SK having the IS1 region and IS2 region, via a
gDCPAL3-derived region sequence (441 bp), in the upstream region of
the 35S promoter region.
(2) Insertion of IS1, IS2, IS12 and MU3 Into a Plant Gene
Expression Vector (FIG. 11)
[0128] pABN-Hm1 (Mita, S., Suzuki-Fujii, K. and Nakamura, K.
Sugar-inducible expression of a gene for .beta.-amylase in
Arabidopsis thaliana. Plant Physiol., 107: 895-904 (1995); gift
from Dr. Kenzo Nakamura at Nagoya Univeristy) was cleaved with
HindIII to thereby excise the .beta.-amylase promoter (1.7 kb),
which was rendered blunt-ended using T4 DNA polymerase, then
cleaved with XbaI, treated with CIP and then subjected to agarose
gel electrophoresis, whereby a 10 kbp DNA fragment containing the
Ti plasmid region as well as the kanamycin resistance gene [nos
promoter/coding region of neomycin phosphotransferase II gene
(nptII)/nos terminator], the coding region (GUS)/nos terminator of
the .beta.-glucuronidase gene, and the hygromycin resistance gene
[35S promoter/coding region (HPT) of hygromycin phosphotransferase
gene/nos terminator] was isolated. In this was subcloned a 35S
fragment obtained from 35S/SK by cleavage with HindIII, rendering
blunt-ended using T4 DNA polymerase and cleaving with XbaI, to
construct pAB35S.
[0129] This was cleaved with XhoI and XbaI and treated with CIP and
then a vector was prepared by cutting off the 35S DNA fragment by
agarose gel electrophoresis. Separately, the IS1-35S/SK,
IS2-35S/SK, IS12-35S/SK and MU3-35S/SK prepared as mentioned above
were each cleaved with XhoI and XbaI and DNA fragments for
insertion (transgene expression cassettes) were recovered by
agarose gel electrophoresis. Such DNA fragments were ligated to the
vector mentioned above and used to transform E. coli DH5 .alpha..
LB agar medium containing 25 .mu.g/L of kanamycin (1%
Bacto-Trypton, 0.5% yeast extract, 1% sodium chloride, 1.5% agar
powder for bacterial culture media) was sowed with each of the E.
coli transformants obtained, and plasmids were extracted from the
colonies obtained by the rapid plasmid DNA extraction method and
the restriction enzyme maps of the plasmids obtained were checked,
whereby the formation of the constructs pIS1-35S/AB35S,
pIS2-35S/AB35S, pIS12-35S/AB35S and pMU3-35S/AB35S, namely the
constructs with the above transgene expression cassettes
respectively inserted between the nptII gene responsible for
kanamycin resistance and the GUS gene, which is a structural gene,
was confirmed as shown in FIG. 11.
(3) Production of Competent Cells of Agrobacterium tumefaciens
[0130] A YEP solid medium (prepared by adding powdered agar for
bacterial culture media to YEP medium comprising 1% yeast extract,
1% Bactoheptone and 0.5% sodium chloride to a concentration of
1.5%, followed by solidification by autoclaving; hereinafter the
same shall apply) was smeared with a loopful of cells taken from a
glycerol stock of A. tumefaciens EHA 101 and the cells were
cultured at 28.degree. C. in the dark for 2 days. Grown single
colonies of A. tumefaciens were each collected with a toothpick and
sowed in 1.5 ml of YEP medium and shake-cultured overnight at
28.degree. C. 80 ml of YEP medium was placed in a 500-ml flask, 0.8
ml of the A. tumefaciens culture fluid was added, and shake culture
was performed at 28.degree. C. until OD.sub.600=0.4. This was
cooled with ice, transferred to a centrifuge tube ice-cooled in
advance, and centrifuged at 6,000 rpm at 4.degree. C. for 5
minutes, the supernatant was removed, and 20 ml of 10% glycerol was
added to suspend the sediment. This procedure was repeated three
times, and the medium was completely removed to give competent
cells of A. tumefaciens. For stocking, the cells were suspended in
400 .mu.l of 10% glycerol and the suspension was distributed in
40-.mu.l portions into tubes, followed by rapid freezing in
liquefied nitrogen.
(4) Introduction of Plasmid DNAs into A. tumefaciens
[0131] The constructs obtained as described above under (2)
(plasmids pIS1-35S/AB35S, pIS2-35S/AB35S, pIS12-35S/AB35S and
pMU3-35S/AB35S) were each introduced into the competent cells of A.
tumefaciens by electoporation (using Shimadzu GTE-10).
Specifically, about 100 ng each of the plasmids prepared as
described above under (2), namely pIS1-35S/AB35S (IS1),
pIS2-35S/AB35S (IS2), pIS12-35S/AB35S (IS12) and pMU3-35S/AB35S
(MU3), and the plasmid pAB35S (35S) to serve as a control with no
insertion of the tanscriptional activation element(s) (IS1 and/or
IS2) was admixed with 40 .mu.l of competent cells prepared as
described above under (3), and each mixture was transferred to an
electroporation cell. Electric pulses (1.2 kV, 35 .mu.F,
550.OMEGA.) were given, and 1 ml of YEP medium was immediately
added, and incubation was performed at 28.degree. C. for 1 hour.
About 50 .mu.l was taken and YEP solid medium containing 50 .mu.g/L
of hygromycin was smeared therewith, and incubation was performed
in the dark at 28.degree. C. for 2 days. A monocolony that had
grown was again spread lightly over another portion of YEP solid
medium containing 50 .mu.g/L of hygromycin using a platinum loop
and incubated at 28.degree. C. for 24 hours. A portion of cells
were taken and planted in 5 ml of YEP medium containing 50 .mu.g/L
of hygromycin and shake-cultured overnight at 28.degree. C.
Example 3
(1) Introduction of Transcriptional Activation Element-Containing
Constructs Into Tobacco Cultured Cells
[0132] Using A. tumefaciens with the construct prepared in Example
2, namely pIS1-35S/AB35S (IS1), pIS2-35S/AB35S (IS2) or
pIS12-35S/AB35S (IS12), introduced therein (hereinafter referred to
as "transformant A. tumefaciens"), the constructs IS1, IS2 and IS12
were respectively introduced into tobacco cultured cells. In a
control run, A. tumefaciens with pAB35S (35S) introduced therein
was used and the same procedure was followed.
[0133] First, the above transformant A. tumefaciens cultured in 5
ml of YEP liquid medium was transferred to a 50-ml centrifuge tube
and centrifuged at 3,000 rpm for 10 minutes. The supernatant was
discarded, 25 ml of Linsmaier & Skoog medium (Linsmaier, E. M.
and Skoog, F.; Physiol. Plantarum 18, 100-127 (1965); hereinafter
referred to as "Lins medium") was added to the sediment and, after
resuspending, centrifugation was carried out again at 3,000 rpm and
at room temperature for 10 minutes, and the supernatant was
discarded. This procedure was repeated four times. Cells of A.
tumefaciens were harvested, Lins medium was added for suspending in
an amount to give OD.sub.600=0.2, acetosyringone was added thereto
to a concentration of 10 .mu.g/ml, followed by resuspending.
[0134] Saparately, cultured tobacco cells BY-2 (gift from Dr.
Toshiyuki Nagata at University of Tokyo) to be used for
introduction of each construct were cultured beforehand in 45 ml of
Lins medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), 1 ml
of the cell-containing suspension culture fluid was transferred to
a fresh portion of Lins medium at one-week intervals, and cells
that had entered the logarithmic growth phase after the lapse of
about 100 hours following transfer were used.
[0135] A sterile 90-mm dish was sowed with said tobacco cultured
cells BY-2 (4 ml), and 100 .mu.l of cells of transformant A.
tumefaciens washed by the above procedure were added uniformly onto
the tobacco cultured cells. After slight blending, co-culture was
carried out in the dark at 22.degree. C. for 3 days.
[0136] Then, 12 ml of Lins medium was added to the cultured cell
fluid for suspending, the suspension was transferred to a 50-ml
centrifuge tube and centrifuged at 1,000 rpm for 1 minute, and the
supernatant was discarded. This procedure was repeated four times.
Then, 12 ml of Lins medium containing 250 .mu.g/ml of claforan was
added and the same procedure as mentioned above was once more
repeated. After discarding the supernatant, about 25 ml of Lins
medium was added to the sediment cells to thereby suspend them, and
the cells in 0.25 .mu.l of the medium were counted using a
hemocytometer. The cells were uniformly sowed onto portions of Lins
solid selection medium containing 100 .mu.g/ml or 300 .mu.g/ml of
kanamycin (further containing 250 .mu.g/ml of claforan) so that the
number of cells per plate amounted to 4.times.10.sup.5,
6.times.10.sup.5, 8.times.10.sup.5, 10.times.10.sup.5 or
15.times.10.sup.5. They were cultured in the dark at 28.degree. C.
After one month of culture, the number of transgenic cultured
tobacco cells (transformant calli) formed on each plate and the
formation rate or yield were determined. The results thus obtained
are shown in Table 1 and FIG. 12. TABLE-US-00001 TABLE 1 Number of
transformant calli formed from cultured tobacco cells BY-2 in
kanamycin-containing medium and rate of transformant callus
formation Number of cells (.times.10.sup.5) 4 6 8 10 Kanamycin 100
.mu.g/ml IS1 4 .+-. 0 20 .+-. 4 43 .+-. 3 99 .+-. 10 (0.10) (0.33)
(0.54) (0.99) IS2 10 .+-. 7 72 .+-. 13 84 .+-. 4 137 .+-. 12 (0.25)
(1.20) (1.05) (1.37) IS12 16 .+-. 3 70 .+-. 6 124 .+-. 16 220 .+-.
4 (0.40) (1.67) (1.55) (2.20) 35S 3 .+-. 2 40 .+-. 8 47 .+-. 2 83
.+-. 13 (Control) (0.08) (0.67) (0.59) (0.83) Kanamycin 300
.mu.g/ml IS1 0 3 .+-. 0 10 .+-. 3 20 .+-. 5 (0.00) (0.05) (0.13)
(0.20) IS2 0 4 .+-. 2 36 .+-. 4 72 .+-. 11 (0.00) (0.07) (0.45)
(0.72) IS12 2 .+-. 1 15 .+-. 1 41 .+-. 2 89 .+-. 5 (0.05) (0.25)
(0.51) (0.89) 35S 0 0 3 .+-. 2 37 .+-. 16 (Control) (0.00) (0.00)
(0.04) (0.37) Upper: Number of calli formed Lower: Yield
(.times.10.sup.-2%)
[0137] From the above results, it was found that insertion of the
construct IS2 or IS12 into tobacco cultured cells increases the
yield of transformant calli in kanamycin-containing medium and that
the yield is higher than the yield of transformant calluses from
cultured tobacco cells (35S) without insertion of such element.
Specifically, the yield of transformant calli (transgenic cultured
tobacco cells) with the construct IS2 or IS12 introduced therein
was 1.6 to 2.6 times higher as compared with the control (35S)
without introduction of such element.
[0138] Particularly when the kanamycin concentration in medium was
300 .mu.g/ml, it was observed that, by introducing the construct
IS2 or IS12, the yield of transformant calli is increased to a
level 10 times or more higher as compared with the control (35S).
This suggests that insertion of the construct IS2 or IS12 result in
an increased level of expression of the ntpII gene responsible for
kanamycin resistance and occurring in the vicinity or marginal
region of said construct.
(2) GUS Activity Measurement in Transformant Tobacco Calli
[0139] Based on the above results, the expression of the GUS gene,
which is a reporter, was checked using the above pIS12-35S/AB35S
(IS12) as the construct, to thereby check whether the above
inserted construct can increase the expression of the structural
gene.
[0140] The expression of the GUS gene was examined by first
randomly selecting independent calli from among a plurality of
transformant tobacco calli, extracting nuclear DNA from each callus
and, after confirming the gene introduction by Southern analysis,
extracting proteins from the callus and measuring the GUS activity.
Specifically, 0.75 g of the transformed tobacco callus was taken
and proteins were extracted using GUS-Light (Tropix, Inc.). The
protein concentration was determined using Bio-Rad's protein assay
kit and then, using GUS-Light (Tropix) and the luminometer Lumat
LB905 (Bertold Japan), the GUS activity was measured for 5 seconds.
The results thus obtained are shown in FIG. 13. In the figure, the
GUS activity (ordinate) is shown in terms of luminescence value per
unit weight of protein as calculated by dividing the luminescence
value obtained from the luminometer by the protein weight.
[0141] As is evident from FIG. 13, the transformant tobacco calli
resulting from insertion of the construct IS12 showed, on an
average, about 2.6 times higher GUS activity as compared with the
control resulting from insertion of element-free pAB35S (35S). From
this, it was found that the expression of the GUS gene is
significantly increased by insertion of the construct IS12.
[0142] This indicates that each element (IS1 element, IS2 element,
and a coupling product therefrom (e.g. IS12 element)) contained in
the above constructs is a transcriptional activation element.
Example 4
Introduction of a Transcriptional Activation Element-Containing
Construct Into a Tobacco Plant (Leaf Disk Method)
[0143] Using A. tumefaciens with the construct prepared in Example
2, namely pIS1-35S/AB35S (IS1), pIS2-35S/AB35S (IS2) or
pIS12-35S/AB35S (IS12), introduced therein (hereinafter referred to
as "transformant A. tumefaciens"), the constructs IS1, IS2 and IS12
were respectively introduced into tobacco leaves by the leaf disk
method. In a control run, A. tumefaciens with the construct-free
pAB35S (35S) introduced therein was used and the same procedure was
followed.
[0144] Specifically, each tobacco (SR 1) leaf was immersed in a 10%
hypochlorous acid solution, air bubbles were removed using a
medicine spoon over 2 minutes with gentle stirring by means of a
stirrer, the solution was renewed and the same procedure was
repeated for further 5 minutes. The leaf was taken out and immersed
in sterilized water, followed by gentle stirring. While replacing
the hypochlorous acid solution with a fresh portion, the same
procedure was repeated three times in all. After removing the
moisture using a sterilized paper towel, the leaf was punched with
a cork borer to give a leaf disk (the vein being removed). This was
immersed in 10 ml of sterilized water. The thus-prepared leaf disk
was immersed in the above-mentioned transformant A. tumefaciens
cultured in 5 ml of YEP medium (adjusted to OD.sub.600=0.25 with
sterilized water). Then, the bacterial suspension and the leaf disk
were together emptied onto a sterilized paper towel and the
moisture was removed with another sterilized paper towel.
[0145] The leaf was placed, inside out, on MS infection medium
prepared by supplementing MS medium (Murashige, T. and Skoog, F.;
Physiol. Plantarum 15, 473-497 (1962)) with 40 mg/L acetosyringone
and 0.2% gelan gum, and cultured in the dark at 25.degree. C. for 2
days. Then, each leaf disk was deprived of bacterial cells in the
manner of wiping with MS differentiation medium (MS medium
supplemented with 0.1 mg/L .alpha.-naphthaleneacetic acid, 1 mg/L
benzyladenine, 150 mg/L kanamycin, 500 mg/L claforan and 0.2% gelan
gum), then placed, inside out, on another portion of MS
differentiation medium and cultured at 25.degree. C. The medium was
replaced with a fresh portion of MS differentiation medium at
two-week intervals and, after the lapse of one month, regenerated
shoots were counted. The results thus obtained are shown in Table
2. TABLE-US-00002 TABLE 2 Comparison in number of regenerated
shoots on tobacco leaf disks Number of shoots per disk 35S IS1 IS2
IS12 0 73 49 56 50 1 26 21 20 23 2 13 17 11 14 3 10 14 8 5 4 3 6 9
8 5 2 1 2 1 6 1 3 3 2 7 0 1 2 5 8 1 0 0 3 9 0 0 0 1 10 0 0 2 1 11 0
0 0 1 12 0 0 0 1 13 0 0 0 0 14 0 0 0 2 Total number of 129 112 113
117 disks Total number of 118 151 164 244 shoots Average number
0.91 1.35 1.45 2.09 of shoots per disk
[0146] As is evident from the above results, the shoot regeneration
efficiency was about 1.4 times when the tobacco plant contained the
construct IS1 or IS2 as compared with the element-free control
(35S) and, in particular when it contained both IS1 and IS2 in a
tandem manner (IS12), about twice as many shoots were obtained as
compared with the control. From this, it is evident that the
elements of the present invention (IS1 element, IS2 element and
coupling products obtained therefrom (IS12 and the like)) can
increase the activity of the kanamycin gene (ntpII gene) occurring
in the vicinity or marginal region of said elements in the tobacco
plant. This result supports the judgment drawn in Example 3 that
the elements of the present invention are transcriptional
activation elements.
Example 5
Introduction of Transcriptional Activation Element-Containing
Constructs into Carrot Somatic Embryos
[0147] Using A. tumefaciens with the construct prepared in Example
2, namely pIS1-35S/AB35S (IS1), pIS2-35S/AB35S (IS2),
pIS12-35S/AB35S (IS12) or pMU3-35S/AB35S (MU3), introduced therein
(hereinafter referred to as "transformant A. tumefaciens"), the
constructs IS1, IS2, IS12 and MU3 were respectively introduced into
carrot somatic embryos. In a control run, A. tumefaciens with the
construct-free pAB35S (35S) introduced therein was used and the
same procedure was followed.
[0148] Specifically, first, carrot hypocotyls germinated under the
sterilized condition were cut to a length of about 1 cm, then
placed in MS medium containing 4.5.times.10.sup.-6 M 2,4-D
(2,4-dichlorophenoxyacetic acid) and cultured in the dark for 24
hours, then placed in 2,4-D-free MS medium and cultured in the dark
for 3 days. The medium was replaced with a fresh portion and
cultivation was performed in the same manner for 7 days to initiate
carrot somatic embryos on the hypocotyls.
[0149] Separately, a culture of the above transformant A.
tumefaciens cultured in 5 ml of YEP medium was centrifuged at 3,000
rpm for 10 minutes, the supernatant was removed, and about 30 ml of
MS medium was added to suspend the cells. This procedure was
repeated twice and the YEP medium was completely removed. Then,
centrifugation was carried out at 3,000 rpm for 10 minutes, the
supernatant was removed, and MS medium containing 10 mg/L of
acetosyringone was added to the sediment to thereby adjust to
OD.sub.600=about 0.3.
[0150] To this were added the above carrot hypocotyls collected
using a net, and the mixture was shaken gently for 5 minutes. The
hypocotyls were deprived of the moisture by wiping with a
sterilized paper towel and immersed in MS medium containing 10 mg/L
of acetosyringone and cultured in the dark at 22.degree. C. for 3
days. The hypocotyls were deprived of the moisture by wiping with a
sterilized paper towel and immersed in MS medium containing 500
.mu.g/L of carbenicillin and washed with the medium by shaking
gently. After removing the moisture in the same manner, the
hypocotyls were cultured in MS agar medium (containing 0.8% agar)
containing 500 .mu.g/L of carbenicillin and 100 .mu.g/L of
kanamycin in the dark. After 1.5 to 3 months, hypocotyls that had
each regenerated a callus were counted. The results thus obtained
are shown in Table 3. TABLE-US-00003 TABLE 3 Comparison in number
of callus-regenerating hypocotyls 35S IS1 IS2 IS12 MU3 +2,4-D
Number of callus-regenerating 5 6 10 9 8 hypocotyls Total number of
78 56 60 61 63 hypocotyls Percent regeneration(%) 6.4 11 17 15 13
-2,4-D Number of callus-regenerating 3 9 12 6 4 hypocotyls Total
number of 77 62 62 64 58 hypocotyls Percent regeneration (%) 3.9 13
19 9.4 6.9
[0151] As is evident from Table 3, with carrot somatic embryos as
well, like the case of tobacco calli, the regeneration efficiency
was found improved upon insertion of the construct IS1, IS2, IS12
and MU3 under the dedifferentiational growth conditions of
culturing in 2,4-D-containing medium (+2,4-D) as well as under the
differentiational growth conditions of culturing in 2,4-D-free
medium (-2,4-D). The greatest improvement in regeneration
efficiency was observed in the case of the construct IS2
inserted.
Example 6
Introduction of Transcriptional Activation Element-Containing
Constructs Into Rice
[0152] Using A. tumefaciens with the construct prepared in Example
2, namely pIS1-35S/AB35S (IS1) or pIS2-35S/AB35S (IS2), introduced
therein (hereinafter referred to as "transformant A. tumefaciens"),
the constructs IS1 and IS2 were respectively introduced into rice
seeds. In a control run, A. tumefaciens with the element-free
pAB35S (35S) introduced therein was used and the same procedure was
followed.
[0153] Specifically, from among fully ripened rice seeds
(Nihonbare), those normal in shape and color, among others, were
first selected and dehulled by lightly rubbing in a mortar.
Dehulled seeds were placed in a 50-ml Falcon tube, 2.5% sodium
hypochlorite was added, and the mixture was shaken at 100-120 rpm
for 20 minutes. Then, the supernatant was discarded, sterilized
water was added, and the mixture was shaken gently. After three
repetitions of this procedure, the seeds were placed on callus
induction medium and cultured in the dark at 28.degree. C.
[0154] After 3 to 4 weeks, among calli formed from scutella and
having growing yellowed shoots, only those having a diameter of 2-3
mm and looking like scattered in a group of several were placed on
fresh callus induction medium and cultured in the dark at
28.degree. C. for 7 days.
[0155] Separately, the above transformant A. tumefaciens was
planted in YEP solid medium and cultured in the dark at 28.degree.
C. for 3 days. Cells of A. tumefaciens were scratched off with a
medicine spoon and added to AAI medium (Toriyama, K. and Hirata,
K.; Plant Science 41, 179-183 (1985)) supplemented with
acetosyringone, and the OD.sub.600 was adjusted to 0.18 to 0.2.
They were shake-cultured in the dark at 25.degree. C. for 1
hour.
[0156] The rice calli cultured in the above manner were placed in a
sterilized tea strainer, and the above cultured A. tumefaciens in
the form of a suspension was added. The tea strainer was shaken for
3 minutes with occasional rocking for securing immersion of the
whole calli, then the tea strainer and the contents were together
placed on a paper towel, and the excessive bacterial culture fluid
was removed. The calli were placed on co-culture medium and
cultured in the dark at 25.degree. C. for 3 days.
[0157] The co-cultured calli were then collected in a tea strainer,
immersed in sterilized water supplemented with 500 mg/l of
claforan, the tea strainer was shaken to wash away A. tumefaciens,
the tea strainer and the contents were then together placed on a
sterilized paper towel, and the water was removed. The same
procedure was repeated four times in all. The calli were placed on
selection medium and cultured in the dark at 28.degree. C. Three to
four weeks later, calli were randomly selected from among a large
number of calli, a portion of each selected callus was taken and
placed in a GUS staining solution (0.75 mM X-Gulc
(5-bromo-4-chloro-3-indolyl-.beta.-D-glucuronic acid), 0.5 mM
potassium ferricyanide, 0.5 mM potassium ferrocyanide, 0.3% Triton
X-100, 20% methanol, 50 mM phosphate buffer (pH 7.0)), and the
reaction was allowed to proceed overnight at 37.degree. C. for
detecting GUS activity. The callus stained blue and thus showing
GUS activity is a transgenic, transformed rice callus. The results
thus obtained are shown in Table 4. TABLE-US-00004 TABLE 4 Results
of rice callus staining for GUS 35S IS1 IS2 Calluses stained 27 38
44 Total number of calluses 275 244 228 Percentage of transformant
calluses (%) 9.8 15.6 19.3
[0158] As is evident from the results shown in Table 4, the
transformation efficiency was found increased by introduction of
the constructs IS1 and IS2 to about 1.5 times and about 2 times,
respectively, as compared with the control (35S).
[0159] The above-mentioned Examples 1 to 6 showed that the
regeneration efficiency (transformation efficiency) is increased by
using the transcriptional activation elements (IS1, IS2, IS12, MU3)
of the present invention. The following three possibilities can be
considered as the reasons: [0160] (1) The possibility of the
efficiency of introduction of Ti plasmid into plant cells being
increased; [0161] (2) The possibility of the efficiency of
regeneration from cells being increased as the result of an
increase in nos promoter activity owing to the IS1 and/or IS2
element and consequent promotion of the transcription of the nptII
gene, which leads to production of the gene product in an increased
amount, and, hence, increase in number of cells capable of growing
on the kanamycin-containing medium used for selection; and [0162]
(3) The possibility of the IS1 and/or IS2 element activating the
gene region in the vicinity or marginal region thereof or
preventing said gene region from being inactivated.
[0163] The gene introduction by means of Ti plasmid does not lead
to insertion at a determined site on the plant chromosome but is
indefinite as to the site of insertion. Therefore, it is accidental
whether the gene in question is inserted in an active site
determined by the structure of the chromosome or in a cryptic site
in the vicinity of which a gene has been inactivated by methylation
of the genomic DNA or by some other cause. It is thought that if a
transgene is introduced in a cryptic site, it is influenced by the
"field" of the chromosome, so that the transgene is also
inactivated.
[0164] In the constructs used in the above examples, the
transcriptional activation elements (IS1 or/and IS2) are found
inserted on the terminator side of the nptII gene (kanamycin
resistance gene), namely on the opposite side of the nos promoter
of the kanamycin resistance gene. Therefore, it is impossible that
these elements cis act on the promoter of the kanamycin resistance
gene.
[0165] However, the above examples gave the results showing that
even in the circumstances in which the transcriptional activation
element of the present invention is found inserted in a position
such that it cannot directly act on the nos promoter, the number of
kanamycin resistant cells (transformant tobacco calli) increases in
the case of cultured tobacco cells, in particular that even when
the kanamycin concentration in medium is as high as 300 mM, the
number of kanamycin resistant calli increases (Example 3 (1)), and
further that the efficiency of regeneration of kanamycin resistant
plants is increased by introducing the constructs mentioned above
into plant cells of various plant species (Examples 4 to 6). These
results indicate that the IS1 or/and IS2 elements acted on the
kanamycin resistance gene occurring in the vicinity of said
elements, not in the mode of directly causing cis activation of the
nos promoter, and as a result, the number of cells retaining
(includes both the senses of activation and prevention from being
inactivated) the activity of said kanamycin resistance gene. Thus,
this indicates the possibility that, unlike the conventional
transcriptional activation elements (factors) occurring as
enhancers in the vicinity of the promoter of a specific gene and
activating the transcription by cis acting on said promoter, the
transcriptional activation element of the present invention is to
activate (or prevent from inactivating), when it is inserted into a
genomic gene, a single gene group or a plurality of gene groups in
the vicinity or marginal region of the site of insertion thereof
(irrespective of location and direction of the gene promoter on
which it acts) and thus promoting the transcription activity,
namely acting by the mechanisms mentioned above under (3).
[0166] In the constructs used in the above examples, the IS1 or/and
IS2 elements were inserted on the 35S promoter side of the GUS
gene. The cultured tobacco cells resulting from insertion of said
construct showed increased GUS activity in Example 3 (2) indicated
clearly that the transcriptional activation elements of the
invention act also on the 35S promoter occurring in the downstream
vicinity thereof to increase the transcription activity of the GUS
gene. This result supports the judgment mentioned above and
indicates that the transcriptional activation elements of the
invention show the actions mentioned above under (3), namely that
"the IS1 or/and IS2 elements activate the neighboring gene region
including them or prevent said gene region from being
inactivated".
[0167] In the above example, it was also shown that, not only with
cultured cells (Example 3) but also with plant tissues, the
efficiency of regeneration of tobacco plant shoots is indeed
increased in the tobacco leaf disk experiment (Example 4), the
efficiency of formation of embryogenic callus serving as bases for
plant regeneration from carrot hypocotyls is increased (Example 5),
and the efficiency of formation of callus serving as bases for rice
plant regeneration is increased (Example 6), by using the
transcriptional activation elements of the invention. These results
indicate that the transcriptional activation elements of the
invention act on those plant cells becoming incapable of plant
regeneration or callus formation as a result of a foreign gene
introduced into the genome of plants in question undergoing gene
silencing (inactivation) by the position effect, so as to increase
the efficiency of plant regeneration or formation, hence are
practically very useful.
[0168] In view of the recent finding that MITEs, like MARs, bind to
nuclear matrices (Tikhonov et al., Plant Cell 12: 249-264 (2000)),
it is considered that MITEs intranuclearly play a role similar to
that of MARs. Based on this, it can be expected that the
transcriptional activation elements of the invention, which are
MITEs, can be used singly or in combination with such elements as
MARs in producing genetically modified plants to further increase
the efficiency of plant regeneration or formation.
INDUSTRIAL APPLICABILITY
[0169] The most important problem to be overcome in producing
genetically modified plants is the phenomenon of gene silencing
which causes expression inactivation of foreign genes. For avoiding
the gene silencing phenomenon in developing genetically modified
plants, it is necessary to select, from among a large number of
plant individuals, those plant individuals with the foreign gene in
question inserted at such a site as causing gene silencing as least
as possible.
[0170] The present invention provides novel, plant-derived
MITE-like elements, and it is highly possible that said MITE-like
elements, when inserted in a plant genome, cause changes in genomic
structure and, based on this, contribute to changes in dynamics of
the genomic structure, for example facilitating the unwinding of
genomic DNA or causing changes in nucleosome structure, by
mechanisms quite different from the mechanisms of action of the
conventional enhancer elements.
[0171] Therefore, by utilizing this characteristic feature of the
MITE-like elements of the invention, it will become possible to
regulate the expression of a gene occurring in the vicinity thereof
by techniques different from the prior art ones. In other words,
with the MITE-like elements of the invention which have the above
characteristic feature, it will be possible to increase or activate
the reduced expression ability of a foreign gene introduced by the
transgenic technology. In this respect, the MITE-like elements of
the invention are useful in constructing a transgene expression
cassette and plasmids containing said cassette in the production of
genetically modified living organisms and is further useful in
stably producing genetically modified organisms capable of
expressing a transgene.
[0172] The transcriptional activation elements of the invention
which contain a transposable element such as one of the MITE-like
elements mentioned above (preferably the IS1 element or/and IS2
element) have an activity in suppressing and dissolving the
phenomenon of inactivation of gene expression (gene silencing
phenomenon) due to the position effect in gene transfer in plants.
Therefore, it is expected that by using the transcriptional
activation elements of the invention singly or in combination with
other elements participating in nuclear DNA structuring, for
example MARs (matrix attachment regions), it will become possible
to have the gene stably expressed to thereby markedly reduce the
number of screening procedures generally performed after gene
transfer and the number of recombinant plants to be sowed and
grown.
[0173] Such plants large in genomic size as lily, chrysanthemum and
wheat have large cryptic sites within the genome and, therefore, a
foreign gene introduced is mostly inserted in cryptic sites. For
such plants, it is thus very difficult and practically impossible
in the prior art to produce recombinant plants. On the contrary,
with the transcriptional activation elements of the invention, it
is possible to significantly inhibit a foreign gene introduced onto
a plant genome from undergoing silencing and therefore it is
expected that said elements can make it possible to efficiently
introduce foreign genes into those plant species the gene
recombination of which has been regarded as difficult, as mentioned
above, and produce recombinant plants.
[0174] The transcriptional activation elements of the invention are
also considered to be elements capable of activating (inclusive of
transcriptional activation) genes occurring in the vicinity or
marginal region of the gene region in which they are found
inserted. Therefore, the transcriptional activation elements of the
invention are expected to not only make it possible to put the
transgenic technology to practical use even in those plants in
which the production of genetically modified plant bodies is
difficult because of frequent occurrence of silencing due to the
large genome size, as mentioned above, but also make it possible,
even in the genetic engineering of plant species such as soybean,
corn, potato and tomato, already put to practical use, to increase
the efficiency of gene transfer and, at the same time, increase the
activity of transcription of genes for useful characters, such as
herbicide resistance genes and insecticidal protein genes, by using
these transcriptional activation elements through insertion at a
site upstream of the promoter of such a gene for a useful
character, or at a site in the vicinity thereof. They are further
expected to make it possible to produce genetically modified plants
with higher productivity and higher quality as compared with the
conventional methods of producing genetically modified plants.
Sequence CWU 1
1
14 1 769 DNA Carrot (Daucus carota L. cv. Kurodagosun) 1 gggatctttt
taaaaatacc catctgtaaa attatttttt taaaaatact accatctttt 60
tcattgtttt taaaaatacc ttttcataaa tttttttttt caaaaatacg atttgcaact
120 tttgcaacct catttgcaac cttgggcggc gcagccgtaa aagttgccag
tgaggttgca 180 aaagttgcaa atgagtttgt aaaagttgca aatgaggttg
caaaagttgc aaataaaaat 240 ggaaagttgc aacagttgca actgcaattg
caactagttc aactgaaaac tgtaagttgc 300 aaaagttgca aatgaggttg
caactaaatg caactgaaaa ctgtaagtaa caacagatgt 360 atggtgtgcc
cctggcgggg ccgttagatt acaatagaat caactgaatg caatcatatg 420
caactgaata caactatatg caatcatata tgcaattaca aatcctgatt tcaagttcca
480 gttttcgaat gtcattttcg aaatcgatat atatatatat atatatatat
cgatttcgaa 540 aatgacattc gaaaactgga acttgaaatc aggaattcag
ctgcatatga agttgcaaaa 600 gaggttgcaa cacggctggc gccgcctgta
gttgcaaatg aggttgcaaa agttgcaaac 660 agtatttttg aaaaaaagat
tttatgaaaa ggtattttta aaaataattc tggaaggtag 720 tatttttgaa
aacaataaaa gaaaaggtag gtagttttgt agatttccc 769 2 299 DNA Carrot
(Daucus carota L. cv. Kurodagosun) 2 ctccctacgt cccattttat
gtgacctcat tttctttttg ggacgtctca aaaaaaataa 60 cctagaatac
ttactatttt ttaacactat ttttcactat tacacccacc aactctatat 120
tttatactat tttattatta aataaacact attacaccca ctacttttct ccactatctc
180 aaatctatta ttaaatattg ataggtccac cactttaccc acttttcaac
tacatttact 240 acatttttct taatctccgt gaaagtcaaa ctcattcaca
taaaatggga cagagggag 299 3 1192 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 3 ggtaccgggc cccccctcga ggtcactccc tacgtcccat
tttatgtgac ctcattttct 60 ttttgggacg tctcaaaaaa aataacctag
aatacttact attttttaac actatttttc 120 actattacac ccaccaactc
tatattttat actattttat tattaaataa acactattac 180 acccactact
tttctccact atctcaaatc tattattaaa tattgatagg tccaccactt 240
tacccacttt tcaactacat ttactacatt tttcttaatc tccgtgaaag tcaaactcat
300 tcacataaaa tgggacagag ggagtaatta ttaattttaa tagacggtat
cgataagctt 360 atcgataccg tctcagattc gcaaacataa aaagaaaagg
gatcttttta aaaataccca 420 tctgtaaaat tattttttta aaaatactac
catctttttc attgttttta aaaatacctt 480 ttcataaatt ttttttttca
aaaatacgat ttgcaacttt tgcaacctca tttgcaacct 540 tgggcggcgc
agccgtaaaa gttgccagtg aggttgcaaa agttgcaaat gagtttgtaa 600
aagttgcaaa tgaggttgca aaagttgcaa ataaaaatgg aaagttgcaa cagttgcaac
660 tgcaattgca actagttcaa ctgaaaactg taagttgcaa aagttgcaaa
tgaggttgca 720 actaaatgca actgaaaact gtaagtaaca acagatgtat
ggtgtgcccc tggcggggcc 780 gttagattac aatagaatca actgaatgca
atcatatgca actgaataca actatatgca 840 atcatatatg caattacaaa
tcctgatttc aagttccagt tttcgaatgt cattttcgaa 900 atcgatatat
atatatatat atatatatcg atttcgaaaa tgacattcga aaactggaac 960
ttgaaatcag gaattcagct gcatatgaag ttgcaaaaga ggttgcaaca cggctggcgc
1020 cgcctgtagt tgcaaatgag gttgcaaaag ttgcaaacag tatttttgaa
aaaaagattt 1080 tatgaaaagg tatttttaaa aataattctg gaaggtagta
tttttgaaaa caataaaaga 1140 aaaggtaggt agttttgtag atttcccaga
cctcgagggg gggcccggta cc 1192 4 8 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 4 gttgcaaa 8 5 8 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 5 gttgcaac 8 6 8 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 6 tttgcaaa 8 7 8 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 7 tttgcaac 8 8 7 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 8 tatgcaa 7 9 7 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 9 aatgcaa 7 10 158 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 10 gggatctttt taaaaatacc catctgtaaa attatttttt
taaaaatact accatctttt 60 tcattgtttt taaaaatacc ttttcataaa
tttttttttt caaaaatacg atttgcaact 120 tttgcaacct catttgcaac
cttgggcggc gcagccgt 158 11 158 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 11 acggctggcg ccgcctgtag ttgcaaatga ggttgcaaaa
gttgcaaaca gtatttttga 60 aaaaaagatt ttatgaaaag gtatttttaa
aaataattct ggaaggtagt atttttgaaa 120 acaataaaag aaaaggtagg
tagttttgta gatttccc 158 12 32 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 12 ctccctacgt cccattttat gtgacctcat tt 32 13 32 DNA
Carrot (Daucus carota L. cv. Kurodagosun) 13 aaactcattc acataaaatg
ggacagaggg ag 32 14 1553 DNA Carrot (Daucus carota L. cv.
Kurodagosun) 14 ggtaccgggc cccccctcga ggtcactccc tacgtcccat
tttatgtgac ctcattttct 60 ttttgggacg tctcaaaaaa aataacctag
aatacttact attttttaac actatttttc 120 actattacac ccaccaactc
tatattttat actattttat tattaaataa acactattac 180 acccactact
tttctccact atctcaaatc tattattaaa tattgatagg tccaccactt 240
tacccacttt tcaactacat ttactacatt tttcttaatc tccgtgaaag tcaaactcat
300 tcacataaaa tgggacagag ggagtaatta ttaattttaa taaattatat
gtgattttga 360 ttatgtgtgg attcttgata aaatatcaca gagttgatat
aaattctaaa gatttaacca 420 caatgtttca aaatctcata gattttagta
ggattcaaaa aatttaaaat acgttcaaaa 480 atctcataaa attcatcatt
ttattaaatc caaaaaaatc cagtaatatt tgacaatcag 540 attttaatga
attttaaatt gaacaatctc agttgaatac catgagattt taaaatataa 600
tttaaaattc taattgaata ccaccagatt ttgtaaaata atttaaaatt ttaattgaat
660 attcaagatt ttaatatatt ttaaataatc tcgatctgaa ttacaaaaaa
tgcttaaaat 720 ctgaatccca tacgtcctct cagattcgca aacataaaaa
gaaaagggat ctttttaaaa 780 atacccatct gtaaaattat ttttttaaaa
atactaccat ctttttcatt gtttttaaaa 840 ataccttttc ataaattttt
tttttcaaaa atacgatttg caacttttgc aacctcattt 900 gcaaccttgg
gcggcgcagc cgtaaaagtt gccagtgagg ttgcaaaagt tgcaaatgag 960
tttgtaaaag ttgcaaatga ggttgcaaaa gttgcaaata aaaatggaaa gttgcaacag
1020 ttgcaactgc aattgcaact agttcaactg aaaactgtaa gttgcaaaag
ttgcaaatga 1080 ggttgcaact aaatgcaact gaaaactgta agtaacaaca
gatgtatggt gtgcccctgg 1140 cggggccgtt agattacaat agaatcaact
gaatgcaatc atatgcaact gaatacaact 1200 atatgcaatc atatatgcaa
ttacaaatcc tgatttcaag ttccagtttt cgaatgtcat 1260 tttcgaaatc
gatatatata tatatatata tatatcgatt tcgaaaatga cattcgaaaa 1320
ctggaacttg aaatcaggaa ttcagctgca tatgaagttg caaaagaggt tgcaacacgg
1380 ctggcgccgc ctgtagttgc aaatgaggtt gcaaaagttg caaacagtat
ttttgaaaaa 1440 aagattttat gaaaaggtat ttttaaaaat aattctggaa
ggtagtattt ttgaaaacaa 1500 taaaagaaaa ggtaggtagt tttgtagatt
tcccagacgg tatcgataag ctt 1553
* * * * *